Herpes simplex virus VP16 vaccines

ABSTRACT

Compositions which are useful for treatment of individuals for Herpes Simplex Virus (HSV) infections are provided, as are methods for their use. These compositions are comprised of immunogenic polypeptides which are comprised of an epitope of HSV VP16; they may also be comprised of an epitope of an HSV glycoprotein. Also provided are polypeptides which are used in the compositions for treating individuals for HSV infection, and methods and compositions used in the production of the polypeptides.

This application is a continuation of application Ser. No. 08/133,974,filed Oct. 8, 1993 now abandoned, which is a continuation of applicationSer. No. 07/561,528 filed Aug. 2, 1990 now abandoned.

TECHNICAL FIELD

This invention relates to materials and methodologies for thealleviation of herpes virus infections. More specifically, it relates tocompositions containing a polypeptide comprised of an immunogenicepitope of VP16, including VP16 and fragments thereof, and to methodsfor preparing the polypeptides for the composition.

BACKGROUND

The herpes viruses include the herpes simplex viruses (HSV), comprisingtwo closely related variants designated types 1 (HSV-1) and 2 (HSV-2).Herpes simplex virus (HSV) is a prevalent cause of genital infection inhumans, with an estimated annual incidence of 600,000 new cases and with10 to 20 million individuals experiencing symptomatic chronic recurrentdisease. The asymptomatic subclinical infection rate may be even higher.Using a type-specific serological assay, researchers showed that 35% ofan unselected population of women attending a health maintenanceorganization clinic in Atlanta had antibodies to HSV type 2 (HSV-2).Although continuous administration of antiviral drugs such as acyclovirameliorates the severity of acute HSV disease and reduces the frequencyand duration of recurrent episodes, such chemotherapeutic interventiondoes not abort the establishment of latency nor does it alter the statusof the latent virus. As a consequence, the recurrent disease pattern israpidly reestablished upon cessation of drug treatment. Since the mainsource of virus transmission arises from recrudescent disease, anyapproach to impact the rate of infection must ultimately require avaccine strategy. Thus, it is a matter of great medical and scientificinterest to provide safe and effective vaccines for humans to preventHSV infection, and where infection has occurred, therapies for thedisease.

HSV is a double stranded DNA virus having a genome of about 150 to 160kbp packaged within an icosahedral capsid surrounded by a membraneenvelope. The viral envelope includes at least seven virus-specificglycoproteins, including gB, gC, gD, gE, and gG, where gB and gD arecross-reactive between types 1 and 2. One approach to vaccine therapyhas been the use of isolated glycoproteins, which have been shown toprovide protection when injected into mice subsequently challenged withlive virus.

The VP16 gene product is associated with the virion tegument, locatedbetween the capsid and the envelope (See FIG. 1). VP16, which is avirion stimulatory factor, is an abundant protein with some 500 to 1000copies per virion. It has been alternately named ICP25, VmW65, and theα-trans-inducing factor (αTIF). The majority of studies on VP16 haveexplored its role in the trans-activation of the "immediate early genes"in HSV replication. In view of the internal location of VP16 in thevirion, and the current state of knowledge concerning the mode of HSVreplication, VP16 would not be expected to be a good candidate for usein treatment of HSV infections.

Relevant Literature

Spear and Roizman (1972) disclose the electrophoretic separation ofproteins in purified HSV1. McLean et al. (1982) discloses a monoclonalantibody which putatively interacts with VP16 from HSV1 and HSV2.

Eberle et al. (1984), discloses studies on antibody response to HSVcomponents during primary and recurrent genital HSV-2 infections.

Campbell et al. (1984), putatively discloses a DNA sequence encodingVmW65 of HSV1, and identifies VmW65 as the major tegument virioncomponent which trans-activates HSV immediate-early (IE) transcription.

Pellett et al. (1985) discloses the expression of cloned HSV1 α-TIFencoding sequence in transient expression systems.

Triezenberg et al. (1988), discloses a putative amino acid sequence forHSV1 VP16, and deletion mutants thereof.

McGeoch et al. (1988) presents a DNA sequence of the long unique region(U_(L)) of HSV-1 strain 17. This region includes a segment whichputatively encodes, in gene UL48, the major tegument protein (which isan activator of transcription of IE genes in the newly infected cell).

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DISCLOSURE OF THE INVENTION

The instant invention results from the discovery that a tegumentpolypeptide of HSV, VP16, is immunogenic and ameliorates the diseasecaused by HSV infection. Thus, the invention includes compositions whichare comprised of an immunogenic epitope of HSV VP16 which are useful forthe treatment of HSV infection, polypeptides used in these compositions,methods of treating HSV infection using these compositions, and methodsof preparing these compositions and immunogenic polypeptides used inthese compositions; also included are vectors comprised ofpolynucleotide sequences encoding these polypeptides, and cellstransformed with the vectors.

Accordingly, one aspect of the invention is a composition for treatmentof an individual for herpes simplex virus (HSV) infection comprising anisolated immunogenic polypeptide containing an immunogenic epitope ofHSV VP16, wherein the polypeptide is present in a pharmacologicallyeffective dose in a pharmaceutically acceptable excipient.

Another aspect of the invention is a composition comprised ofrecombinant vaccinia virus, wherein the virus is comprised of a sequenceencoding an immunogenic polypeptide selected from HSV VP16, truncatedHSV VP16, and mutants thereof, wherein the polynucleotide encoding theimmunogenic polypeptide is operably linked to a control sequence.

Yet another aspect of the invention is a method of producing acomposition for treatment of HSV infection comprising:

(a) providing an immunogenic polypeptide comprised of an immunogenicepitope of HSV VP16;

(b) formulating the polypeptide in a pharmaceutically acceptableexcipient.

Another aspect of the invention is a composition produced by the abovemethod.

Still another aspect of the invention is a method of treating anindividual for HSV infection comprising administering to the individualthe above-described compositions.

An additional aspect of the invention is a recombinant polynucleotideencoding a polypeptide comprised of an immunogenic epitope of HSV-2VP16.

Yet another aspect of the invention is a recombinant vector comprised ofthe above-described polynucleotide.

Yet another aspect of the invention is a recombinant expression systemcomprising an open reading frame (ORF) of DNA encoding a polypeptidecomprised of an immunogenic epitope of HSV-2 VP16, wherein the ORF isoperably linked to a control sequence compatible with a desired host.

Another aspect of the invention is a host cell transformed with therecombinant expression system of claim 38.

Still another aspect of the invention is a method of producing animmunogenic polypeptide for use in the treatment of HSV infection, themethod comprising:

(a) providing the above-described host cell;

(b) incubating the host cell under conditions which allow expression ofthe polypeptide; and

(c) isolating the expressed polypeptide from the host cell.

Still another aspect of the invention is an immunogenic polypeptide foruse in the treatment of HSV infection, produced by the above-describedmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of an HSV virion.

FIG. 2 shows the putative amino acid sequences of HSV-1 VP16 and HSV-2VP16.

FIGS. 3-A and 3-B shows the nucleotide sequence encoding HSV-2 VP16, andthe amino acids encoded therein.

FIG. 4 is a map showing some significant features of the vector pAC373,of pVL985, and the sequence encoding the n-terminal amino acids of thepolyhedrin gene.

FIG. 5 is a map showing some significant features of the vector pHS225.

FIGS. 6A-E is a copy of FIG. 4 of WO88/02634, which presents thenucleotide sequence encoding HSV gB2, and the amino acids encodedtherein.

FIG. 7 is a map showing some significant features of the vector pHS218.

FIG. 8 is a map showing some significant features of the vector pBCB07.

FIG. 9 is a map showing some significant features of the vectorpVACC-gB2.

FIG. 10 is a schematic showing the contents of wells in an antibodytiter study.

FIG. 11 is a bar graph showing the titer of HSV specific complementdependent neutralizing antibody titers resulting from immunization withvv-gB2 and vv-VP16.

FIG. 12 is a graph showing the time course of protection resulting fromimmunization with vv-gB2.

FIG. 13 is a graph showing the time course of protection resulting fromimmunization with vv-VP16.

FIG. 14 is a graph showing the time course of protection resulting fromimmunization with vv-gB2, vv-VP16, and vv-gB2+vv-VP16.

MODES FOR CARRYING OUT THE INVENTION

The following terminology is used herein.

The term "polypeptide" refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

The term "isolated polypeptide" refers to a polypeptide which issubstantially free of other HSV vital components, particularlypolynucleotides. A polypeptide composition is "substantially free" ofanother component if the weight of the polypeptide in the composition isat least 70% of the weight of the polypeptide and other componentcombined, more preferably at least about 80%, still more preferablyabout 90%, and most preferably 95% or greater. For example, acomposition containing 100 μg/mL VP16 and only 3 μg/mL HSV components(e.g., DNA, lipids, etc.) is substantially free of "other HSV viralcomponents," and thus is a composition of an isolated polypeptide withinthe scope of this definition. Similarly, some compositions of theinvention comprise an isolated VP16 polypeptide in combination with oneor more isolated HSV glycoproteins, e.g., gB, gC, gD, and the like.

A "recombinant polynucleotide" intends a polynucleotide of genomic,cDNA, semisynthetic, or synthetic origin which, by virtue of its originor manipulation: (1) is not associated with all or a portion of apolynucleotide with which it is associated in nature, (2) is linked to apolynucleotide other than that to which it is linked in nature, or (3)does not occur in nature.

A "polynucleotide" is a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. This term refers only tothe primary structure of the molecule. Thus, this term includes double-and single-stranded DNA and RNA. It also includes known types ofmodifications, for example, labels which are known in the art,methylation, "caps", substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g.,metals,radioactive metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.), as well asunmodified forms of the polynucleotide.

"Recombinant host cells", "host cells", "cells", "cell lines", "cellcultures", and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be or have been, used as recipients for a recombinant vectoror other transfer polynucleotide, and include the progeny of theoriginal cell which has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

A "replicon" is any genetic element, e.g., a plasmid, a chromosome, avirus, a cosmid, etc., that behaves as an autonomous unit ofpolynucleotide replication within a cell; i.e., capable of replicationunder its own control.

A "vector" is a replicon further comprising sequences providingreplication and/or expression of the open reading frame.

"Control sequence" refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and terminators; in eukaryotes,generally, such control sequences include promoters, terminators and, insome instances, enhancers. The term "control sequences" is intended toinclude, at a minimum, all components whose presence is necessary forexpression, and may also include additional components whose presence isadvantageous, for example, leader sequences which govern secretion.

A "promoter" is a nucleotide sequence which is comprised of consensussequences which allow the binding of RNA polymerase to the DNA templatein a manner such that mRNA production inititiates at the normaltranscription initiation site for the adjacent structural gene.

"Operably linked" refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence "operably linked" to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequencewhich encodes a polypeptide; this region may represent a portion of acoding sequence or a total coding sequence.

A "coding sequence" is a polynucleotide sequence which is transcribedinto mRNA and/or translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5'-terminus and a translation stop codon at the 3'-terminus. A codingsequence can include but is not limited to mRNA, DNA (including cDNA),and recombinant polynucleotide sequences.

As used herein, "epitope" refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation which is unique to the epitope. Generally an epitopeconsists of at least 5 such amino acids, and more usually, consists ofat about 8 to 10 such amino acids. Methods of determining the spatialconformation of such amino acids are known in the art, and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticreference.

An "immunogenic epitope" is an epitope in a polypeptide that elicits acellular and/or humoral immune response; the response may be elicited bythe polypeptide alone, or may require the presence of a carrier in thepresence or absence of an adjuvant.

An epitope is the "immunologic equivalent" of another epitope in adesignated polypeptide when it has the amino acid sequence andconformation which allows it to cross-react with antibodies which bindimmunologically to the epitope in the designated polypeptide.

As used herein, an epitope of a designated polypeptide denotes epitopeswith the same amino acid sequence as the epitope in the designatedpolypeptide, and immunologic equivalents thereof.

A polypeptide which is "comprised of an immunogenic epitope of HSV VP16"is a polypeptide which contains a sequence of amino acids of HSV VP16 ofat least the number to form the immunogenic epitope, usually at leastabout five amino acids, more usually at least about 8 amino acids, andeven more usually about 10 or more amino acids; the maximum size is notcritical. The amino acid sequence from HSV VP16 may be linked at theamino terminus and/or carboxy terminus to another polypeptide (e.g., acarrier protein), either by covalent attachment or by expressing a fusedpolynucleotide to form a fusion protein. If desired, one may insert orattach multiple repeats of the epitope, and/or incorporate a variety ofepitopes. The carrier protein may be derived from any source, but willgenerally be a relatively large, immunogenic protein such as BSA, KLH,or the like. If desired, one may employ a substantially full-length VP16protein as the carrier, multiplying the number of immunogenic epitopes.Alternatively, the amino acid sequence from HSV VP16 may be linked atthe amino terminus and/or carboxy terminus to a non-HSV VP16 amino acidsequence, thus the polypeptide would be a "fusion polypeptide".Analogous types of polypeptides may be constructed using epitopes fromother designated viral proteins.

A "mutant" of a designated polypeptide refers to a polypeptide in whichthe amino acid sequence of the designated polypeptide has been alteredby the deletion or substitution of one or more amino acids in thesequence, or by the addition of one or more amino acids to the sequence.Methods by which mutants occur (for example, by recombination) or aremade (for example, by site directed mutagenesis) are known in the art.

"Transformation" refers to the insertion of an exogenous polynucleotideinto a host cell, irrespective of the method used for the insertion, forexample, direct uptake, transduction (including viral infection),f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid or viralgenome, or alternatively, may be integrated into the host genome.

An "individual" refers to a vertebrate, particularly a member of amammalian species, and includes but is not limited to domestic animals,sports animals, and primates, including humans.

As used herein, "treatment" refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantialelimination of the virus. Treatment may be effected prophylactically(before or prior to infection) or therapeutically (during or followinginfection).

The term "effective amount" refers to an amount of epitope-bearingpolypeptide sufficient to induce an immune response in the subject towhich it is administered. The immune response may comprise, withoutlimitation, induction of cellular and/or humoral immunity. Preferably,the effective amount is sufficient to effect treatment, as definedabove. The exact amount necessary will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the particular polypeptideselected and its mode of administration, etc. Thus, it is not possibleto specify an exact effective amount. However, the appropriate effectiveamount may be determined by one of ordinary skill in the art using onlyroutine experimentation.

The term "HSV glycoprotein" refers to any of the glycoproteins found inthe membrane region of HSV-1, HSV-2, and related herpes viruses.Presently preferred HSV glycoproteins are gB, gC, gD, and gE. Includedwithin this definition are glycoproteins extracted from natural viruses(e.g., from infected sera or cell culture) and glycoproteins produced byrecombinant methods. Such glycoproteins may be modified, either bychemical or enzymatic means (e.g., by proteolytic cleavage,deglycosylation, etc.), or by mutation, or by recombinant DNA techniques(e.g., by fusing HSV glycoprotein genes with other genes to providefusion proteins, or by deleting or replacing sections of DNA sequence).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, Maniatis, & Fitsch, MOLECULAR CLONING, A LABORATORY MANUAL,Second Edition (1989); DNA CLONING, VOLUMES I and II (D. N. Glover, Ed.1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed. (1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELLCULTURE (R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRLPress, 1986; B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984);the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.), andparticularly Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds.,respectively); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Millerand M. P. Calos eds. 1987, Cold Spring Harbor Laboratory),IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press,London), Scopes, (1987); PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE,Second Edition (Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Volumes I-IV, (D. M. Weir and C. C. Blackwell, eds., 1986.)All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated herein by reference.

Compositions of the invention, which are used to treat individuals forHSV infection, are comprised of a polypeptide which contains one or moreimmunogenic epitopes of HSV VP16. The surprising result that HSV VP16 isimmunogenic, and protective, is demonstrated in Examples 4 and 5, infra.Thus, the compositions comprised of polypeptides containing at least oneimmunogenic epitope of HSV VP16, may be used for treatment ofindividuals to prevent or lessen the disease symptoms associated withHSV infections. Moreover, the results also show that the addition of anHSV glycoprotein to the vaccine which is comprised of HSV VP16 enhancesboth the immunogenic and protective effect. Therefore, in a preferredmode, the vaccines are further comprised of at least one immunogenicepitope of an HSV glycoprotein. The glycoprotein epitope may exist onthe same polypeptide as the VP16 epitope, or may exist on a secondpolypeptide. In a more preferred mode, the glycoprotein epitope is fromHSV gB or HSV gD.

In order to prepare the vaccine, a polypeptide comprised of one or moreimmunogenic epitopes of HSV VP16 is provided. If an immunogenic epitopeof an HSV glycoprotein is also desired in the vaccine it may also beincluded in the polypeptide comprised of the HSV VP16 epitope, oralternatively, it may be provided in a second polypeptide.

The provided polypeptides may be full-length HSV VP16 and/or HSVglycoproteins. If the provided polypeptides are full length, they may beisolated from the virus. Isolation and further purification may beaccomplished by techniques known in the art. See, for example, Methodsin Enzymology, and Scopes, PROTEIN PURIFICATION, which discuss a varietyof methods for purifying proteins.

Alternatively, the full length polypeptides may be synthesized usingrecombinant DNA techniques and either the known sequences which encodethe glycoproteins and the HSV-1 VP16, or the sequence for HSV-2 VP16provided herein. The full length polypeptides may contain one or moresubstitutions in the amino acid sequence, as long as the immunogenicityof the designated polypeptide is still evident.

The invention also contemplates the use of polypeptides comprised oftruncated HSV VP16 and/or glycoprotein amino acid sequences. The size ofpolypeptides comprising the truncated HSV VP16 sequences or glycoproteinsequences can vary widely, the minimum size being a sequence ofsufficient size to provide the desired immunogenic epitope, whilemaximum size is not critical. For convenience, the maximum size usuallyis not substantially greater than that required to provide the desiredepitopes and function(s) of the heterologous sequence, if any.Typically, the truncated HSV amino acid sequence will range from about 5to about 400 amino acids in length. More typically, however, the viralsequence containing the immunogenic epitope will be a maximum of about100 amino acids in length, preferably a maximum of about 50 amino acids.

Truncated HSV VP16 or HSV glycoprotein amino acid sequences which areimmunogenic can be identified in a number of ways. For example, theentire viral protein sequence can be screened by preparing a series ofshort peptides that together span the entire protein sequence. Bystarting with, for example, 100 mer polypeptides, it would be routine totest each polypeptide for the presence of epitope(s) showing a desiredreactivity, and then testing progressively smaller and overlappingfragments from an identified 100 mer to map the epitope of interest.Screening such peptides in an immunoassay is within the skill of theart, and appropriate immunoassays for immunogenicity are described inthe Examples. Methods of computer analysis of a protein sequence toidentify potential epitopes are also known. For example, putativeepitopes of HSV-2 VP16 have been determined from the putative amino acidsequence shown in FIG. 2, using as criteria the surface probability,antigen index, hydrophilicity, charge, or lack of overt structure of theregions of the HSV-2 VP16 polypeptide. These putative epitopes arelocated at about amino acid (aa) 15 to about aa 34; at about aa 193 toabout aa 220; at about aa 320 to about aa 330; at about aa 360 to aboutaa 371; at about aa 378 to about aa 390; at about aa 400 to about aa410; and at about aa 480 to about aa 490. After the identification ofputative epitopes, oligopeptides comprising the identified regions canbe prepared for screening.

If desired, a single polypeptide may include at least one truncated HSVVP16 sequence which includes an immunogenic epitope, and also, at leastone truncated HSV glycoprotein sequence which includes an immunogenicepitope. Alternatively, the truncated HSV VP16 and HSV glycoproteinsequences may be on separate polypeptides. While truncated sequences canbe produced by various known treatments of the subject native viralprotein(s), it is generally preferred to make synthetic or recombinantpolypeptides comprised of the desired immunogenic epitopes.

Recombinant polypeptides comprised of the truncated HSV VP16 sequencescan be made up entirely of VP16 sequences (one or more epitopes, eithercontiguous or noncontiguous), or VP16 sequence or sequences in a fusionprotein. Similarly, polypeptides comprised of truncated HSV glycoproteinsequences can be made up entirely of the glycoprotein sequence (one ormore epitopes, either contiguous or noncontiguous), or the glycoproteinsequence or sequences in a fusion protein.

In fusion proteins, useful heterologous sequences include sequences thatprovide for secretion from a recombinant host, enhance the immunologicalreactivity of the VP16 or glycoprotein epitope(s), or facilitate thecoupling of the polypeptide to a support or a vaccine carrier. See,e.g., EPO Pub. No. 116,201; U.S. Pat. No. 4,722840; EPO Pub. No.259,149; U.S. Pat. No. 4,629,783, the disclosures of which areincorporated herein by reference.

Full length as well as polypeptides comprised of truncated HSV VP16and/or HSV glycoprotein sequences, and mutants thereof, may be preparedby recombinant technology. A DNA sequence putatively encoding HSV-1 VP16(also known as VmW65) is disclosed in Campbell et al (1984), thedisclosure of which is incorporated herein by reference. A DNA sequenceencoding HSV-2 VP16, discovered by the herein inventors and described inExample 1, is provided in FIG. 3, infra. In the figure, the Metindicated by the arrow is the putative initiating methionine. The methodfor the provision of the sequence of HSV-2 VP16 is simply of historicalinterest, since the information in the sequence data is available bothin FIG. 3 and in ATCC Deposit No. 68,372, which is incorporated hereinby reference. The sequences encoding a number of HSV glycoproteins,including gB and gD are known. For example, sequences encoding HSV-1 andHSV-2 gB are shown in U.S. Pat. No. 4,642,333; sequences encoding HSV gDare described in Watson et al. (1982). Methods for expressing gB and gD,and fragments thereof, are described in WO88/02634. The availability ofthese sequences permits the construction of polynucleotides encodingimmunogenic regions of the HSV VP16 polypeptides and HSV glycoproteins.

Polynucleotides encoding the desired polypeptide comprised of one ormore of the immunogenic HSV VP16 epitopes and/or one or more of theimmunogenic glycoprotein epitopes may be chemically synthesized orisolated, and inserted into an expression vector. The vectors may or maynot contain portions of fusion sequences such as beta-Galactosidase orsuperoxide dismutase (SOD). Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in European Patent Office Publication number 0196056,published Oct. 1, 1986.

The DNA encoding the desired polypeptide, whether in fused or matureform and whether or not containing a signal sequence to permitsecretion, may be ligated into expression vectors suitable for anyconvenient host. The hosts are then transformed with the expressionvector. Both eukaryotic and prokaryotic host systems are presently usedin forming recombinant polypeptides, and a summary of some of the morecommon control systems and host cell lines is presented infra. The hostcells are incubated under conditions which allow expression of thedesired polypeptide. The polypeptide is then isolated from lysed cellsor from the culture medium and purified to the extent needed for itsintended use.

The general techniques used in extracting the genome from a virus,preparing and probing DNA libraries, sequencing clones, constructingexpression vectors, transforming cells, performing immunological assayssuch as radioimmunoassays and ELISA assays, for growing cells inculture, and the like, are known in the art and laboratory manuals areavailable describing these techniques. However, as a general guide, thefollowing sets forth some sources currently available for suchprocedures, and for materials useful in carrying them out.

Synthetic oligonucleotides may be prepared using an automatedoligonucleotide synthesizer as described by Warner (1984). If desired,the synthetic strands may be labeled with ³² P by treatment withpolynucleotide kinase in the presence of ³² P-ATP, using standardconditions for the reaction.

In order to create mutants, or to create desired functional sequences orto remove them, (e.g., restriction enzyme sites) DNA sequences,including those isolated from clones, may be modified by knowntechniques, including for example, site directed mutagenesis, asdescribed by Zoller (1982). Briefly, the DNA to be modified is packagedinto phage as a single stranded sequence, and converted to a doublestranded DNA with DNA polymerase using, as a primer, a syntheticoligonucleotide complementary to the portion of the DNA to be modified,and having the desired modification included in its own sequence. Theresulting double stranded DNA is transformed into a phage supportinghost bacterium. Cultures of the transformed bacteria, which containreplications of each strand of the page, are plated in agar to obtainplaques. Theoretically, 50% of the new plaques contain phage having themutated sequence, and the remaining 50% have the original sequence.Replicates of the plaques are hybridized to labeled synthetic probe attemperatures and conditions which permit hybridization with the correctstrand, but not with the unmodified sequence. The sequences which havebeen identified by hybridization are recovered and cloned.

Generally, in hybridization analysis, the DNA to be probed isimmobilized on nitrocellulose filters, denatured, and prehybridized witha buffer containing 0-50% formamide, 0.75M NaCl 75 mM Na citrate, 0.02%(wt/v) each of bovine serum albumin, polyvinyl pyrollidone, and Ficoll,50 mM Na phosphate (pH 6.5), 0.1% SDS, and 100 μg/ml carrier denaturedDNA. The percentage of formamide in the buffer, as well as the time andtemperature conditions of the prehybridization and subsequenthybridization steps and wash depends on the stringency required.Oligomeric probes which require lower stringency conditions aregenerally used with low percentages of formamide, lower temperatures,and longer hybridization times. Probes containing more than 30 or 40nucleotides such as those derived from cloned DNAs generally employhigher temperatures, e.g., about 40°-42° C., and a high percentage,e.g., 50% formamide. Following prehybridization, labeled probe is addedto the buffer, and the filters are incubated in this mixture underhybridization conditions. After washing, the treated filters aresubjected to autoradiography to show the location of the hybridizedprobe; DNA in corresponding locations on the original agar plates isused as the source of the desired DNA.

Vector construction employs techniques which are known in the art.Site-specific DNA cleavage is performed by treating with suitablerestriction enzymes under conditions which generally are specified bythe manufacturer of these commercially available enzymes. In general,about 1 μg of plasmid or DNA sequence is cleaved by 1 unit of enzyme inabout 20 μl buffer solution by incubation of 1-2 hr at 37°C. Afterincubation with the restriction enzyme, protein is removed by extraction(e.g., with phenol/chloroform), and the DNA recovered (e.g., byprecipitation with ethanol). The cleaved fragments may be separated,e.g., using gel electrophoresis techniques or by sedimentation,according to the general procedures found in Methods in Enzymology(1980) 65:499-560.

Sticky ended cleavage fragments may be blunt ended using E. coli DNApolymerase I (Klenow) in the presence of the appropriate deoxynucleotidetriphosphates (dNTPs) present in the mixture. Treatment with a singlestranded nuclease (e.g., S1 nuclease) may also be used to hydrolyze anysingle stranded DNA portions.

Ligations may be carried out using standard buffer and temperatureconditions using T4DNA ligase and ATP. When vector fragments are used aspart of a ligation mixture, the vector fragment is often treated withbacterial alkaline phosphatase (BAP) or calf intestinal alkalinephosphatase to remove the 5'-phosphate and thus prevent religation ofthe vector; alternatively, restriction enzyme digestion of unwantedfragments can be used to prevent ligation.

Ligation mixtures are used to transform suitable cloning hosts which areknown in the art, e.g., E. coli, and successful transformants areselected by an appropriate marker, for example, antibiotic resistance,and screened for the correct construction.

In order to verify constructions, ligation mixtures are transformed intoa suitable host, e.g., E. coli HB101, and successful transformantsselected by antibiotic resistance or other markers. Plasmids from thetransformants are then prepared according to the method of Clewell etal. (1969), usually following chloramphenicol amplification (Clewell(1972)). The DNA is isolated and analyzed, usually by restriction enzymeanalysis and/or sequencing. Sequencing may be by the dideoxy method ofSanger et al. (1977), as further described by Messing et al. (1981), orby the method of Maxam et al. (1980). Problems with band compression,which are sometimes observed in GC rich regions, may be overcome by useof T-deazoguanosine according to Barr et al. (1986).

Transformation of the vector containing the desired sequence into theappropriate host may be by any known method for introducingpolynucleotides into a host cell, including, for example, packaging thepolynucleotide in a virus and transducing the host cell with the virus,or by direct uptake of the polynucleotide. The transformation procedureused depends upon the host to be transformed. For example, in vivotransformation using vaccinia virus as the transforming agent forpolynucleotides encoding HSV-2 VP16 is described infra., in theExamples. Transformation may also be accomplished in vitro systems.Bacterial transformation by direct uptake generally employs treatmentwith calcium or rubidium chloride (Cohen (1972); Sambrook (1989)). Yeasttransformation by direct uptake may be carried out using the method ofHinnen et al. (1978). Mammalian transformations by direct uptake may beconducted using the calcium phosphate precipitation method of Graham andVan der Eb (1978), or the various known modifications thereof. Othermethods for the introduction of recombinant polynucleotides into cells,particularly into mammalian cells, which are known in the art includedextran mediated transfection, calcium phosphate mediated transfection,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the polynucleotides into nuclei.

In order to obtain expression of desired coding sequences, host cellsare transformed with polynucleotides (which may be expression vectors),which are comprised of control sequences operably linked to the desiredcoding sequences. The control sequences are compatible with thedesignated host. Among prokaryotic hosts, E. coli is most frequentlyused. Expression control sequences for prokaryotes include promoters,optionally containing operator portions, and ribosome binding sites.Transfer vectors compatible with prokaryotic hosts are commonly derivedfrom, for example, pBR322, a plasmid containing operons conferringampicillin and tetracycline resistance, and the various pUC vectors,which also contain sequences conferring antibiotic resistance markers.Promoter sequences may be naturally occurring, for example, theβ-lactamase (penicillinase) (Weissman (1981)), lactose (lac)(Chang etal. (1977), and tryptophan (trp)(Goeddel et al. (1980)), andlambda-derived P_(L) promoter system and N gene ribosome binding site(Shimatake et al. (1981)). In addition, synthetic promoters which do notoccur in nature also function as bacterial promoters. For example,transcription activation sequences of one promoter may be joined withthe operon sequences of another promoter, creating a synthetic hybridpromoter (e.g., the tac promoter, which is derived from sequences of thetrp and lac promoters (De Boer et al. (1983)). The foregoing systems areparticularly compatible with E. coli; if desired, other prokaryotichosts such as strains of Bacillus or Pseudomonas may be used, withcorresponding control sequences.

Eukaryotic hosts include yeast and mammalian cells in culture systems.Saccharomyces cerevisiae and Saccharomyces carlsbergensis are the mostcommonly used yeast hosts, and are convenient fungal hosts. Yeastcompatible vectors carry markers which permit selection of successfultransformants by conferring prototrophy to auxotrophic mutants orresistance to heavy metals on wild-type strains. Yeast compatiblevectors may employ the 2 micron origin of replication (Broach et al.(1983)), the combination of CEN3 and ARS1 or other means for assuringreplication, such as sequences which will result in incorporation of anappropriate fragment into the host cell genome. Control sequences foryeast vectors are known in the art and include promoters for thesynthesis of glycolytic enzymes (Hess et al. (1968)); for example,alcohol dehydrogenase (ADH)(E.P.O. Publication No. 284044), enolase,glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphatedehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase,3-glycerophosphate mutase, and pyruvate kinase (PyK)(E.P.O. PublicationNo. 329203). The yeast PHO5 gene, encoding acid phosphatase, alsoprovides useful promoter sequences (Miyanohara et al. (1983). Inaddition, synthetic promoters which do not occur in nature also functionas yeast promoters. For example, upstream activating sequences (UAS) ofone yeast promoter may be joined with the transcription activationregion of another yeast promoter, creating a synthetic hybrid promoter.Examples of such hybrid promoters include the ADH regulatory sequencelinked to the GAP transcription activation region (U.S. Pat. Nos.4,876,197 and 4,880,734). Other examples of hybrid promoters includepromoters which consist of the regulatory sequences of either the ADH2,GAL4, GAL10, or PHO5 genes, combined with the transcriptional activationregion of a glycolytic enzyme gene such as GAP or PyK (E.P.O.Publication No. 164556). Furthermore, a yeast promoter can includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase for the appropriate inititiation oftranscription.

Other control elements which may be included in the yeast expressionvector are terminators (e.g., from GAPDH, and from the enolase gene(Holland (1981)), and leader sequences. The leader sequence fragmenttypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell. DNA encodingsuitable signal sequences can be derived from genes for secreted yeastproteins, such as the yeast invertase gene (E.P.O. Publication No.12,873) and the α-factor gene (U.S. Pat. No. 4,588,684). Alternatively,leaders of non-yeast origin, such as an interferon leader, also providefor secretion in yeast (E.P.O. Publication No. 60057). A preferred classof secretion leaders are those that employ a fragment of the yeastα-factor gene, which contains both a "pre" signal sequence, and a "pro"region. The types of α-factor fragments that can be employed include thefull-length pre-pro α-factor leader, as well as truncated α-factorleaders (U.S. Pat. Nos. 4,546,083 and 4,870,008; E.P.O. Publication No.324274. Additional leaders employing an α-factor leader fragment thatprovides for secretion include hybrid α-factor leaders made with apre-sequence of a first yeast, but a pro- region from a second yeastα-factor. (See, e.g., P.C.T. WO 89/02463).

Expression vectors, either extrachromosomal replicons or integratingvectors, have been developed for transformation into many yeasts. Forexample, expression vectors have been developed for Candida albicans(Kurtz et al. (1986)), Candida maltosa (Kunze et al. (1985)), Hanzenulapolymorpha (Gleeson et al. (1986)), Kluyveromyces fragilis (Das et al.(1984)), Kluyveromyces lactis (De Louvencourt et al. (1983)), Pichiaguillerimondii, (Kunze et al. (1985)), Pichia pastoris (Cregg et al.(1985); U.S. Pat. Nos. 4,837,148 and 4,929,555)), Schizosaccharomycespombe (Beach and Nurse (1981)), and Yarrowia lipolytica (Davidow et al.(1985)).

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including, for example, HeLa cells,Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, COSmonkey cells, and a number of other cell lines. Suitable promoters formammalian cells are also known in the art and include viral promoterssuch as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV),adenovirus (ADV) and bovine papilloma virus (BPV) (See, Sambrook (1989)for examples of suitable promoters). Mammalian cells may also requireterminator sequences and poly A addition sequences; enhancer sequenceswhich increase expression may also be included, and sequences whichcause amplification of the gene may also be desirable. These sequencesare known in the art.

Vectors suitable for replication in mammalian cells are known in theart, and may include viral replicons, or sequences which ensureintegration of the appropriate sequences encoding the desiredpolypeptides into the host genome.

A vector which is used to express foreign DNA and which may be used invaccine preparation is Vaccinia virus. In this case, the heterologousDNA is inserted into the Vaccinia genome. Techniques for the insertionof foreign DNA into the vaccinia virus genome are known in the art, andutilize, for example, homologous recombination. The insertion of theheterologous DNA is generally into a gene which is non-essential innature, for example, the thymidine kinase gene (tk), which also providesa selectable marker. Plasmid vectors that greatly facilitate theconstruction of recombinant viruses have been described (see, forexample, Mackett et al. (1984), Chakrabarti et al. (1985); Moss (1987)).Expression of the desired polypeptides comprised of immunogenic regionsthen occurs in cells or individuals which are infected and/or immunizedwith the live recombinant vaccinia virus.

Other systems for expression of polypeptides include insect cells andvectors suitable for use in these cells. These systems are known in theart, and include, for example, insect expression transfer vectorsderived from the baculovirus Autographa californica nuclear polyhedrosisvirus (AcNPV), which is a helper-independent, viral expression vector.Expression vectors derived from this system usually use the strong viralpolyhedrin gene promoter to drive expression of heterologous genes.Currently the most commonly used transfer vector for introducing foreigngenes into AcNPV is pAc373, shown in FIG. 4. Many other vectors, knownto those of skill in the art, have also been designed for improvedexpression. These include, for example, pVL985 (which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 basepairs downstream from the ATT; See Luckow andSummers (1989). AcNPV transfer vectors for high level expression ofnonfused foreign proteins are shown in FIG. 4. In the figure, thenumbers shown refer to positions within the native gene, where the A ofthe ATG codon is +1. FIG. 4 also shows a restriction endonuclease map ofthe transfer vector pAc373. The map shows that a unique BamHI site islocated following position -8 with respect to the translation initiationcodon ATG of the polyhedrin gene. There are no cleavage sites for SmaI,PstI, BglI, XbaI or SstI. Good expression of nonfused foreign proteinsusually requires foreign genes that ideally have a short leader sequencecontaining suitable translation initiation signals preceding an ATGstart signal. The plasmid also contains the polyhedrin polyadenylationsignal and the ampicillin-resistance (amp) gene and origin ofreplication for selection and propagation in E. coli.

Methods for the introduction of heterologous DNA into the desired sitein the baculovirus are known in the art. (See Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555; Ju et al. (1987);Smith et al. (1983); and Luckow and Summers (1989)). For example, theinsertion can be into a gene such as the polyhedrin gene, by homologousrecombination; insertion can also be into a restriction enzyme siteengineered into the desired baculovirus gene. The inserted sequences maybe those which encode all or varying segments of HSV VP16 and/or HSVglycoprotein.

The signals for posttranslational modifications, such as signal peptidecleavage, proteolytic cleavage, and phosphorylation, appear to berecognized by insect cells. The signals required for secretion andnuclear accumulation also appear to be conserved between theinvertebrate and vertebrate cells. Examples of the signal sequences fromvertebrate cells which are effective in invertebrate cells are known inthe art, for example, the human interleukin 2 signal (IL2_(s)) which isa signal for transport out if the cell, is recognized and properlyremoved in insect cells.

It is often desirable that the polypeptides prepared using the abovehost cells and vectors be fusion polypeptides. As with non-fusionpolypeptides, fusion polypeptides may remain intracellular afterexpression. Alternatively, fusion proteins can also be secreted from thecell into the growth medium if they are comprised of a leader sequencefragment. Preferably, there are processing sites between the leaderfragment and the remainder of the foreign gene that can be cleavedeither in vivo or in vitro.

In instances wherein the synthesized polypeptide is correctly configuredso as to provide the correct epitope, but is too small to beimmunogenic, the polypeptide may be linked to a suitable carrier. Anumber of techniques for obtaining such linkage are known in the art,including the formation of disulfide linkages usingN-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP) and succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptidelacks a sulfhydryl group, this can be provided by addition of a cysteineresidue.) These reagents create a disulfide linkage between themselvesand peptide cysteine resides on one protein and an amide linkage throughthe ε-amino on a lysine, or other free amino group in other amino acids.A variety of such disulfide/amide-forming agents are known. See, forexample, Immun. Rev. (1982) 62:185. Other bifunctional coupling agentsfor a thioether rather than a disulfide linkage. Many of thesethio-ether-forming agents are commercially available and includereactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid,2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid,and the like. The carboxyl groups can be activated by combining themwith succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.Additional methods of coupling antigens employ the rotavirus/"bindingpeptide" system described in EPO Publication No. 259,149. The foregoinglist is not meant to be exhaustive, and modifications of the namedcompounds can clearly be used.

Any carrier may be used which does not itself induce the production ofantibodies harmful to the host. Suitable carriers are typically large,slowly metabolized macromolecules such as proteins; polysaccharides suchas latex functionalized sepharose, agarose, cellulose, cellulose beadsand the like; polymeric amino acids, such as polyglutamic acid,polylysine, and the like; amino acid copolymers; and inactive virusparticles (see infra.). Especially useful protein substrates are serumalbumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well knownto those of skill in the art.

The immunogenicity of the epitopes of HSV VP16, particularly of HSV-2VP16, and of HSV glycoproteins, particularly HSV gB and/or HSV gD, mayalso be enhanced by preparing them in eukaryotic systems fused with orassembled with particle-forming proteins such as, for example, thatassociated with hepatitis B surface antigen. See, e.g., U.S. Pat. No.4,722,840. Constructs wherein the HSV VP16 or glycoprotein epitope islinked directly to the particle-forming protein coding sequencesproduces hybrids which are immunogenic with respect to the HSV epitope.In addition, all of the vectors prepared include epitopes specific toHBV, having various degrees of immunogenicity, such as, for example, thepre-S peptide. Thus, particles constructed from particle forming proteinwhich include HSV sequences are immunogenic with respect to HSV and HBV.

Hepatitis surface antigen (HBSAg) has been shown to be formed andassembled into particles in S. cerevisiae (Valenzuela et al. (1982), aswell as in, for example, mammalian cells (Valenzuela et al. (1984)). Theformation of such particles has been shown to enhance the immunogenicityof the monomer subunit. The constructs may also include theimmunodominant epitope of HBSAg, comprising the 55 amino acids of thepresurface (pre-S) region. Neurath et al. (1984). Constructs of thepre-S-HBSAg particle expressible in yeast are disclosed in E.P.O.Publication No. 174,444; hybrids including heterologous viral sequencesfor yeast expression are disclosed in E.P.O. Publication No. 175,261.These constructs may also be expressed in mammalian cells such as CHOcells using an SV40-dihydrofolate reductase vector (Michelle et al.(1984)).

In addition, portions of the particle-forming protein coding sequencemay be replaced with codons encoding an HSV VP16 or HSV glycoproteinepitope. In this replacement, regions which are not required to mediatethe aggregation of the units to form immunogenic particles in yeast ormammals can be deleted, thus eliminating additional HBV antigenic sitesfrom competition with the HSV epitope(s).

The preparation of vaccines which contain an immunogenic polypeptide(s)as an active ingredient(s) is known to one skilled in the art.Typically, such vaccines are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. thepreparation may also be emulsified, or the polypeptide(s) encapsulatedin liposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include, but are notlimited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE, and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against an immunogenicpolypeptide containing an HSV-VP16 epitope and/or HSV glycoproteinepitope, the antibodies resulting from administration of thispolypeptide in vaccines which are also comprised of the variousadjuvants.

The proteins may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or organic acids such as acetic, oxalic, tartaric, maleic, and the like.Salts formed with the free carboxyl groups may also be derived frominorganic bases such as, for example, sodium potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides;such suppositories may beformed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

In addition to the above, it is also possible to prepare live vaccinesof attenuated microorganisms which express one or more recombinantpolypeptides comprised of HSV VP16 and/or HSV glycoprotein epitopes.Suitable attenuated microorganisms are known in the art and include, forexample, viruses (e.g., vaccinia virus) as well as bacteria.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective. The quantity to be administered, which isgenerally in the range of 5 μg to 250 μg of antigen per dose, depends onthe subject to be treated, capacity of the subject's immune system tosynthesize antibodies, and the degree of protection desired. Preciseamounts of active ingredient required to be administered may depend onthe judgment of the practitioner and may be peculiar to each individual.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand/or reenforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at lest in part, be determined by the needof the individual and be dependent upon the judgment of thepractitioner.

In addition, the vaccine containing the polypeptide comprised of animmunogenic HSV VP16 epitope may be administered in conjunction withother immunoregulatory agents, for example, immune globulins.

EXAMPLES Example 1

Isolation and Sequencing of a Gene Encoding HSV-2 VP16

The EcoRI "L" fragment of HSV-2 strain G was inserted into pUC19 toyield pH2G512, the source of the polynucleotide which encodes HSV-2VP16. The HSV-2 polynucleotide encoding sequence was identified bySouthern blot analysis, using as probe a segment of pRB3458, whichcontains the sequence which encodes HSV-1 VP16. The plasmids pH2G512 andpRB3458 were obtained from Dr. P. Pellett (Center for Disease Control,Atlanta, Ga.) and Dr. B. Roizman (University of Chicago, Chicago, Ill.),respectively. The construction of pRB3458 is described in Pellet et al.(1985).

More specifically, the HSV-2 VP16 encoding polynucleotide, pH2G512 wasdigested with EcoRI, and EcoRI and SacI, SacII, BamHI, NcoI, and SmaI,respectively. The fragments of the digested plasmid were separated byelectrophoresis on a 1% agarose gel in trisacetate buffer. Afterelectrophoresis, the DNA in the gel was denatured with alkali,neutralized, and transferred to a "Gene Screen Plus" membrane (DupontNEN), using the transfer protocol described in Sambrook et al. (1989).The DNA on the membrane was hybridized with the probe overnight usingthe manufacturer's directions; the probe was a nick-translated 2.9 KbEcoRI-HindIII fragment isolated from pRB3458. Results of thehybridization showed that HSV-2 VP16 is encoded in a 3.5 Kb EcoRI-SacIfragment of pH2G512. Subsequently, the VP16 encoding EcoRI-SacI fragmentwas isolated on a 1% agarose gel, and extracted from the gel using "GeneClean" (Bio 101). Sequencing of the fragment was accomplished by thedideoxy method. Since HSV DNA is G-C rich (i.e., >70% G-C), sequences inareas of compressions in the sequencing gels were resolved by sequencingwith Taq polymerase at 65° C. The sequence of the coding strand of thefragment, and the amino acids encoded therein, are shown in FIG. 3. Inthe figure, the first nucleotide of the putative initiating methioninecodon is shown by an arrow.

Homologies between the putative amino acid sequences for HSV-1 VP16 andHSV-2 VP16 are shown in FIG. 2.

Example 2

Construction of a Vaccinia Virus Expression Vector Comprised of aSequence Encoding HSV-2 VP16

A vaccinia vector comprised of a sequence encoding HSV-2 VP16 wasconstructed as follows. Initially the VP16 encoding sequence wassubcloned into the vaccinia expression vector, pSC11, to generateplasmid pHS225 (a partial map of which is shown in FIG. 5. The vectorpSC11 was obtained from Dr. Bernard Moss, National Institutes of Health,Bethesda, Md. Prior to introduction of the HSV-2 VP16 coding sequence,the pSC11 vector was modified by deletion of the HindIII site in pSC11by digestion with HindIII, followed by treatment with the Klenowfragment of DNA polymerase I, and ligation. The vector was then furthermodified by the introducing into the SmaI site, a polylinker containingrestriction enzyme sites for SmaI, KpnI, BglII, and HindIII. Thefragment containing VP16 was isolated as a 2.1 Kb XhoI-SphI fragmentfrom pH2G512. The Xho site was filled in using the Klenow fragment ofDNA polymerase I, and the SphI site was blunted using T4 DNA polymerase.The blunt-ended fragment was then ligated into the SmaI site of themodified pSC11. Vectors containing the VP16 encoding sequence wereobtained by cloning; they were transformed into DH5α and transformantswere selected using Ap^(r) selection; positive clones were selectedbased on the presence of the appropriate size fragment after restrictionenzyme analysis. One of the positive clones was named pHS225. A mapshowing some of the significant features of pHS225 is shown in FIG. 5.

In order to obtain a recombinant vaccinia virus vector which wassuitable for expressing VP16 in individuals, the VP16 encoding sequenceof pHS225 was inserted into the TK locus of wild type vaccinia strain,WR, by recombination using the Lipofectin™ (GIBCO BRL, Gaithersburg,Md.) transfection protocol described by the manufacturer of Lipofectin™.Recombinant TK⁻ viruses were isolated by BuDR selection, andplaque-purified using the protocol of Mackett et al. (1987). Avaccinia/VP16 recombinant clone was selected by DNA dot blothybridization. Expression of VP16 was verified by Western Blot andradioimmunoprecipitation, and the recombinant clone was subsequentlypurified. The details of this procedure are as follows.

In order to obtain recombinants of pHS225 with vaccinia WR, confluentmonolayers of BSC40 cells in T-25 flasks were infected with WR at amultiplicity of infection (moi) of 0.05; adsorption was performed fortwo hours at room temperature with rocking. Three pHS225 solutions wereprepared with Lipofectin™ as follows: 50 μl of a DNA solution containingeither 1, 10, or 100 μg pHS225 in water were mixed with 30 μgLipofectin™ plus 20 μl water. The solutions were allowed to incubate atroom temperature for 15 minutes. The infected cells were washed twice inserum free medium; 3 ml of serum free medium was added to each flask;then 100 μl of a DNA-Lipofectin™ complex was added dropwise to eachflask with swirling. Transfections were incubated at 37° in anatmosphere containing 7% CO₂ for 5 hours. Then 3 mls of DME containing20% fetal calf serum (FCS) was added to each flask (final FCSconcentration was 10%), and the transfections were incubated for 48-72hours. After the incubation, recombinant virus was harvested by scrapingthe cells into the medium. Virus was released from the cells byfreeze-thawing the cells three times.

Recombinant viruses containing VP16 were selected using the technique ofMackett et al. (1987). Briefly, the virus stock generated by eachtransfection was thawed, sonicated and incubated 30 min. at 37° C. inthe presence of 0.1 volume of 0.25% trypsin. Monolayers of TK-143 cellswere infected with 10-fold serial dilutions of the trypsinized stock.After adsorption, the cells were overlaid with DME containing 1% lowmelting point agarose, 5% FCS and 25 μg BUDR (Sigma Chemical Co.). At 48hours post infection (p.i.), the cells were stained with 1% agarosecontaining 0.1% neutral red. After 3 to 5 hours, viral plaques werevisualized as clearings in the cell lawn. Plaques were picked, andsubjected to two more rounds of plaque purification using BUDR.

Verification that the selected recombinants contained the VP16 encodingsequence was accomplished by dot blot hybridization. The dot blottechnique was essentially according to the technique of Mackett et al.(1987), except that detection was with a fragment encoding VP16.Briefly, cells infected with putative recombinants were dotted ontonitrocellulose using a dot blot manifold, lysed and denatured. Filterswere baked at 80° C. in vacuo for 2 hours, treated before hybridizationwith a solution containing 60% formamide, 1% sodium dodecyl sulfate(SDS), 1M NaCl, 10% dextran sulfate, and hybridized with 10⁶ cpm/ml of³² p! labeled VP16. Hybridizations were carried out overnight at 42° C.in a solution containing 60% formamide, 1% sodium dodecyl sulfate (SDS),1M NaCl, 10% dextran sulfate, 10 mg/ml salmon sperm DNA, 10 mg/ml polyA⁺ DNA and 50 mg/ml yeast tRNA. After hybridization, the filters werewashed four times with 2×SSC for 5 minutes at room temperature, oncewith 2×SSC, 0.1% SDS for 30 minutes at 65° C., and once with 0.1×SSC,0.1% SDS for 30 minutes at 65° C. The results of the hybridizationshowed that 6 of 12 isolates were positive for the HSV-2 VP16 codingsequence, and that 2 of the 12 isolates were strongly positive. Sixisolates prepared as described above were chosen for further analysis ofthe expression of VP16.

Example 3

Expression of VP16 from Recombinant vv-VP16 Vectors

Expression of VP16 from the vv-VP16 clones described in Example 2 wasdetected by radioimmunoprecipitation of ³⁵ S!-labeled infected celllysates using high-titer positive human sera followed bySDS-polyacrylamide gel electrophoresis of the precipitated products, andsubsequent visualization of ³⁵ S!-labeled-VP16 by autoradiography. Morespecifically, the samples were electrophoresed on 8%, 1.5 mm thicknesspolyacrylamide gels (Novex Corp.) for 90 minutes at 40 mA. Afterelectrophoresis the gels were fixed, "enhanced" and dried prior toexposure to film. The apparent molecular weight of the recombinant VP16(Vmw65) is 65 kD. The identity of VP16 was confirmed byradioimmunoprecipitation of protein from HSV-2 infected Vero cells,using the VP16 specific monoclonal antibody, LP1, for the precipitation.LP1, which is described in McLean et al. (1982), was obtained from A.Minson, Cambridge University. The labeled precipitated product from thevv-VP16 infected cells co-migrated during electrophoresis with thelabeled precipitated product from the Vero cells. This co-migrationduring electrophoresis of the VP16 expressed in Vero cells and from therecombinant vaccinia virus-VP16 (vv-VP16) cells indicate that thevv-VP16 product is full length.

It is of interest that the antibody LP1 does not recognize VP16expressed in the vv-VP16 cells, whereas it does recognize VP16 expressedin HSV infected Vero cells. It is possible that the change in antibodyrecognition in the vv-VP16 product results from a lack ofphosphorylation of the recombinantly produced polypeptide, or otherdifferences in protein processing, since vaccinia virus replicates inthe cytoplasm of the infected cell.

Example 4

Immunogenicity and Protective Effect of Immunization with VP16 or gB2

In order to compare the effect of immunization with VP16 to that withgB2, with respect to their immunogenicity and protection against HSV-2caused disease, vaccinia virus recombinants encoding each polypeptidewere used to immunize guinea pigs. The vaccinia recombinant used whichcontains the gene coding for VP16 was that described in Example 2, i.e.,vv-VP16 (also called vv-VP16-TK⁻). The gB recombinant was prepared bysubcloning a polynucleotide encoding gB2 into a pUC13 vector. The gB2encoding polynucleotide, which was a 3.2 Kb HindIII-BamHI fragment,contained nucleotides from position-136 to 3088, as shown in FIG. 4 inWO88/02634; the latter figure is included herein as FIG. 6. Significantfeatures of the resulting vector, pHS218, are shown in FIG. 7. In orderto produce a vaccinia virus expression vector encoding gB2, a 3.2 KbHindIII-BamHI fragment excised from pHS218 was blunt ended, and ligatedinto the HincII site of pCB07 yielding the vector, pVACC-gB2⁻.Significant features of the vectors pCB07 and pVACC-gB2 are shown inFIG. 8 and FIG. 9, respectively. Similar to the vv-VP16 construct, thisplaces the vaccinia promoter, 7.5, upstream of the gene; the flankingthymidine kinase (TK) sequences provide for recombination into the wildtype virus at this locus. The construction of pVACC-gB2 from pHS218 wasperformed by Dr. Ian Ramshaw, The John Curtin School of MedicalResearch, The Australian National University, Canberra, Australia. Theprocedures for the production of the vaccinia expression vector, vv-gB2,from wild-type vaccinia virus were similar to those for the productionof vv-VP16, except that recombination was with pVACC-gB2, and selectionfor positive clones was by hybridization with a radiolabeled fragmentencoding gB2.

Female guinea pigs were immunized either intradermally (by scarificationof the skin below the right intercostal margin with a bifurcatedneedle), interperitoneally, or intravenously (into an ear vein using a30 gauge needle). The protocol for each of the groups in the study areshown in the following Table 1.

                  TABLE 1                                                         ______________________________________                                        Immunization with vv-gB2 or vv-VP16                                           Group    Route of Immunization                                                                         Immunizations I & II                                 ______________________________________                                        1        I.D.            10.sup.8 pfu vv-gB2                                  2        I.P.            10.sup.8 pfu vv-gB2                                  3        I.V.            10.sup.8 pfu vv-gB2                                  4        I.D.            10.sup.8 pfu vv-VP16                                 5        I.P.            10.sup.8 pfu vv-VP16                                 6        I.V.            10.sup.8 pfu vv-VP16                                 ______________________________________                                    

The animals were immunized twice with a one-month interval betweenimmunizations. The animals were bled for the determination ofHSV-specific and vaccinia-specific neutralizing antibodies at 3 and 6weeks following the second immunization. Animals in groups 1 through 6were challenged with 3×10⁵ pfu of HSV-2 strain MS; challenge was on day64, 6 weeks after the second immunization boost. Challenge was byintravaginal inoculation of HSV-2. The animals were scored for acutedisease the first 14 days post-challenge.

In order to measure the immunogenicity of VP16 and gB2, the titers ofneutralizing antibodies resulting from the immunizations, bothcomplement dependent and complement independent, were determined asfollows. A suspension of 150 μl of Vero cells (1.1×10⁶ cells per 15 mlmedium containing 10% fetal calf serum (FCS)) were seeded in two 96 wellflat bottom plates ("Microtest III" Tissue Culture plates from Falcon),and incubated overnight in a CO₂ incubator at 37° C. On the next day,samples were prepared in a third 96 well plate, the well contents wereas shown in FIG. 10. In the figure, the medium was DME-H21 tissueculture medium, heat-inactivated fetal calf serum (HI FCS) was preparedby incubating FCS (Hyclone Corp.) at 56° C. for 30 min., the guinea pigcomplement was a 1:125 dilution of rehydrated guinea pig complement(Gibco Corp., prepared according to the manufacturer's directions). Afourth plate was also prepared, which was analogous to the third plate,but in which the guinea pig complement was omitted. The plates wereincubated for 2 hours. Viral absorption and replication was accomplishedby aspirating the culture medium from the cell monolayers in plates 1and 2. The contents of the corresponding wells in plates 3 and 4 weretransferred to plates 1 and 2, respectively, and the plates weremaintained in a CO₂ incubator at 37° C. for three days. In order todetect cell cytolysis due to viral replication, after the three dayincubation, the culture medium was aspirated from the cells, 100 μl of aphosphate buffered saline solution containing 10% formaldehyde solutionand 0.09% crystal violet was added to each well. The plates wereincubated 15 min at room temperature, the crystal violet solution wasremoved, and the wells were washed three times with water and the plateswere air dried. The viral titers were 3(2^(n)) and 2(2^(n)) for thecomplement dependent and complement independent samples, respectively; nequals the serum dilution that inhibits cytolysis of the cell monolayerby 50%.

The results on the antibody titrations, expressed as the meanneutralizing titers found in bleeds 1 and 2 for HSV-specific complementdependent neutralizing antibody titers are shown in FIG. 11. As seen inthe figure, at 3 weeks (bleed 1), I.V. administration of w-VP16increased the complement dependent neutralizing antibodies approximatelyfive-fold higher than did vv-gB2.

The HSV-specific complement independent neutralizing antibody titers forbleed 1 are shown in the following Table 2. As seen from the Table 2,I.V. administration of vv-VP16 yielded titers of antibodies whichexceeded HSV-specific titers induced by the vv-gB2 recombinant by >10fold. Neutralization was determined as 50% reduction in plaqueformation. Animals immunized with wild-type non-recombinant vaccinia,WR, do not elicit measurable neutralizing antibodies.

                  TABLE 2                                                         ______________________________________                                        HSV-Specific Complement-Independent                                           Neutralizing Antibody Titers                                                  Group     Vaccine    Administration                                                                           Titer 2.sub.3 *                               ______________________________________                                        1         vv-gB2     I.D.       32 ± 0                                     2         vv-gB2     I.P.       32 ± 0                                     3         vv-gB2     I.V.       32 ± 0                                     4         vv-VP16    I.D.       32 ± 0                                     5         vv-VP16    I.P.       40 ± 8                                     6         vv-VP16    I.V.       565 ± 53                                   ______________________________________                                         *Titer 2.sub.3 signifies the average titer at 3 weeks.                   

The effect of immunization with vv-VP16 and vv-gB2 on protection asreflected in lesions and the severity of acute disease were alsocompared. The clinical course of primary genital HSV-2 infection isgenerally as follows. Lesions first appear on the external genitalia ofall animals three to four days after viral inoculation. The lesionsbegin as discrete vesicles with an erythematous base, and rapidlyprogress to multiple vesiculo-ulcerative lesions by days 5-8.Hemorrhagic crusts cover the ulcerative lesions by days 8-10. Loss ofthe crusts with complete healing of the external genital skin occurs bydays 13-15. Most animals develop urinary retention between day 5 and day10; however, this symptom is resolved by days 10-15. Hindlimb paralysismay be evident in 0% to 20% of animals by days 7-10; this symptom isresolved by days 15-20. Infection and external genital lesions occur in80-100% of the inoculated animals with death rates of 0-50%. The lesionscoring for the studies was according to the following scale:

0.5=redness, swelling;

1.0=1-2 vesicles, or 1 vesicle accompanied by redness and swelling;

1.5=2-4 small vesicles (1-2 mm diameter) or 2 vesicles accompanied byswelling and redness;

2.0=4-6 vesicles

2.5=4-6 large vesicles (greater than 2 mm diameter) with swelling andredness;

3.0=greater than 6 large vesicles;

3.5=greater than 6 large vesicles accompanied by additional smallervesicles;

4.0=confluent vesicles covering greater than one-half of the perineum;

4.5=extreme vesicles with ulceration.

The results comparing the effect of the immunization with vv-VP16 tovv-gB2 on protection against the disease as indicated by the occurrenceand severity of lesions as well as on mortality, are shown in thefollowing Table 3. In the study all animals developed lesions; thelesion score for the unimmunized control group was 3.10.

                  TABLE 3                                                         ______________________________________                                        Effect of Immunization with vv-gB2 or vv-VP16                                 on the Clinical Course of Acute Genital                                       HSV-2 Infection                                                                                       Lesion                                                Group  Vaccine   Route  Score    Protection                                                                           Mortality                             ______________________________________                                        1      vv-gB2    I.D.   1.89 ± .19                                                                          39%    1/4                                   2      vv-gB2    I.P.   1.33 ± .15                                                                          57%    0                                     3      vv-gB2    I.V.   1.29 ± .13                                                                          58%    0                                     4      vv-VP16   I.D.   2.85 ± .16                                                                           8%    1/4                                   5      vv-VP16   I.P.   2.33 ± .17                                                                          25%    0                                     6      vv-VP16   I.V.   1.62 ± .14                                                                          48%    0                                     ______________________________________                                    

The time course of protection with the different routes of immunizationwith vv-gB2 and vv-VP16 are shown in FIG. 12 and FIG. 13, respectively.

Example 5

Immunogenicity and Protective Effect of Immunization with VP16 and gB2

The immunogenicity and protective effect of vv-VP16 and vv-gB2 againstHSV-2 caused disease was examined using a protocol similar to that inExample 4, except that the administration of the vaccines was I.V., andthe challenge dose with HSV-2 strain MS was 6×10⁴ pfu. The vaccines wereadministered to four groups as follows: group 1, no treatment; group 2,vv-VP16+vv-gB2; group 3, vv-gB2 alone; and group 4, vv-VP16 alone.

The results of the study on the production of complement-dependentneutralizing antibody titers is shown in the following Table 3. In thestudy, neutralization was determined as 50% reduction in plaqueformation. Animals immunized with vaccinia WR do not elicit measurableneutralizing antibodies (<16). These results indicate that, at threeweeks post-immunization, treatment with vaccine comprised of bothvv-VP16 and vv-gB2 caused higher titers of neutralizing antibodies thandid either vv-gB2 or vv-VP16 alone; at six weeks immunization withvv-VP16 appeared to be almost equivalent to that with vv-VP16 and vv-gB2with respect to the antibody titers.

                  TABLE 4                                                         ______________________________________                                        Effect of vv-VP16 and vv-gB2                                                  on Complement Dependent                                                       Neutralizing Antibody Titers                                                                         Mean Titer                                                                              Mean Titer                                   Group    Vaccine       (3 weeks) (6 weeks)                                    ______________________________________                                        1        None          13 ± 6 14 ± 2                                    2        vv-VP16 + vv-gB2                                                                            590 ± 57                                                                             500 ± 61                                  3        vv-gB2        113 ± 12                                                                             224 ± 34                                  4        vv-VP16       213 ± 39                                                                             615 ± 61                                  ______________________________________                                    

The protective effect of the combined vaccine comprised of vv-VP16 andvv-gB2 relative to the single subunit vaccines was also monitored, usingthe procedures (with the above modifications) and scoring described inExample 4. In the study, all of the animals exhibited lesions. However,the results, shown in the following Table 5, indicated that the acutedisease was ameliorated by the vaccines, and that the protective effectof the combination vaccine was enhanced relative to either vv-gB2 orvv-VP16 alone. The time course of the protective effect is shown in FIG.14.

                  TABLE 5                                                         ______________________________________                                        The Effect of vv-VP16 and vv-gB2 on                                           Acute Genital HSV-2 Infection                                                                   Lesion                                                      Group    Vaccine  Score      Protection                                                                           Mortality                                 ______________________________________                                        1        None     2.27 ± .28                                                                            --      4/8 (50%)                                2        vv-vPl6  0.59 ± .09                                                                            74%    0/8 (0%)                                           + vv-gB2                                                             3        vv-gB2   1.12 ± .14                                                                            50.7%  0/8 (0%)                                  4        vv-VP16  1.05 ± .11                                                                            53.8%  0/8 (0%)                                  ______________________________________                                    

The following listed materials are on deposit under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC), 12301Parklawn Dr., Rockville, Md. 20852, and have been assigned the followingAccession Numbers

    ______________________________________                                        Material         Deposit Date                                                                            ATCC No.                                           ______________________________________                                        pHS226 in E. Coli DH5α                                                                   15 July 1990                                                                            68372                                              ______________________________________                                    

Upon allowance and issuance of this application as a United StatesPatent, all restriction on availability of these deposits will beirrevocably removed; and access to the designated deposits will beavailable during pendency of the above-named application to onedetermined by the Commissioner to be entitled thereto under 37 CFR 1.14and 35 USC 1.22. Moreover, the designated deposits will be maintainedfor a period of thirty (30) years from the date of deposit, or for five(5) years after the last request for the deposit; or for the enforceablelife of the U.S. patent, whichever is longer. The deposited materialsmentioned herein are intended for convenience only, and are not requiredto practice the present invention in view of the descriptions herein,and in addition these materials are incorporated herein by reference.

INDUSTRIAL APPLICABILITY

The compositions described herein, which contain an immunogenicpolypeptide comprised of an epitope of HSV VP16, are useful for thealleviation of symptoms resulting from herpes simplex virus infections.The recombinant vectors, expression systems, and host cells transformedby these vectors are useful for the preparation of the immunogenicpolypeptides, which in turn are useful in the preparation of the abovedescribed vaccines.

What is claimed is:
 1. A method of producing a composition for treatmentand/or prevention of HSV infection comprising:(a) providing an isolatedimmunogenic polypeptide of HSV VP16 capable of eliciting a cellularimmune response, said polypeptide comprising at least about the first400 amino acids of HSV-2 VP16; (b) formulating the polypeptide in apharmaceutically acceptable excipient; and (c) providing an adjuvant. 2.The method of claim 1, wherein the polypeptide is selected from thegroup consisting of HSV-2 VP16 and a truncated HSV-2 VP16 comprising atleast about the first 400 amino acids of HSV-2 VP16 but less than allthe amino acids of full-length HSV-2 VP16.
 3. The method of claim 1,further comprising providing a second isolated immunogenic polypeptidewhich comprises an immunogenic epitope of an HSV glycoprotein selectedfrom the group consisting of gB and gD.
 4. The method of claim 3,wherein the second polypeptide is selected from the group consisting ofan HSV gB, an HSV gD, a truncated HSV gB and a truncated HSV gD.
 5. Themethod of claim 3, wherein the second polypeptide is HSV gB.
 6. Themethod of claim 3, wherein the second polypeptide is HSV gD.
 7. Themethod of claim 3, further comprising providing a third isolatedimmunogenic polypeptide which comprises an immunogenic epitope of asecond HSV glycoprotein selected from the group consisting of gB and gD,with the proviso that when the second polypeptide is a gB polypeptide,the third polypeptide is a gD polypeptide and when the secondpolypeptide is a gD polypeptide, the third polypeptide is a gBpolypeptide.
 8. The method of claim 7, wherein the second polypeptide isHSV gD and the third polypeptide is HSV gB.
 9. The method of claim 7,wherein the second polypeptide is HSV gB and the third polypeptide isHSV gD.
 10. The method of claim 7, wherein the third polypeptide isselected from the group consisting of an HSV gB, an HSV gD, a truncatedHSV gB and a truncated HSV gD.
 11. A virus comprising a recombinantpolynucleotide which comprises an open reading frame (ORF) of DNAencoding an immunogenic polypeptide of HSV-2 VP16 capable of eliciting acellular immune response, said polypeptide comprising at least about thefirst 400 amino acids of HSV-2 VP16.
 12. The virus of claim 11, whereinthe ORF encodes an immunogenic polypeptide selected from the groupconsisting of HSV-2 VP16and a truncated HSV-2 VP16 comprising at leastabout the first 400 amino acids of HSV VP16-2 but less than all theamino acids of full-length HSV-2 VP16.
 13. The virus of claim 11,wherein the virus is a vaccinia virus.